The Bunyaviridae is the largest family of RNA viruses, comprising five genera (Orthobunyavirus, Hantavirus, Phlebovirus, Nairovirus and Tospovirus) and containing more than 350 viruses. Members of the Bunyaviridae are found worldwide and their host range is very broad, including arthropods, mammals (including humans and bats), birds and even plants. Furthermore, viruses belonging to this family cause a number of important human diseases e.g., La Crosse encephalitis, hantavirus pulmonary syndrome, Rift Valley fever, Crimean-Congo hemorrhagic fever). Many of these viruses are listed as NIAID category A, B or C pathogens for priority research on molecular/cellular virology, pathogenesis and the development of countermeasures. Bunyaviruses possess a tri-segmented negative-stranded RNA genome consisting of large (L), medium (M) and small (S) genome segments. For the genus Orthobunyavirus, the L segment encodes the RNA-dependent RNA polymerase (L) protein, while the M segment encodes the glycoproteins Gn and Gc, as well as the non-structural protein NSm, and the S segment encodes both the nucleocapsid protein (N) and the non-structural protein NSs. The evolution of bunyaviruses is complex, being driven by antigenic drift (accumulation of mutations), antigenic shift (segment reassortment) and virus/host interactions, making it difficult to understand the mechanisms of biogenesis and emergence/re-emergence of highly pathogenic viruses. In particular, genetic reassortment between different bunyaviruses is believed to be a major driving force in the evolution of bunyaviruses, but in contrast to other segmented RNA viruses (e.g. influenza virus) detailed molecular biological and epidemiological/ecological studies regarding the mechanisms underlying the acquisition of virulence and emergence of new bunyavirus have not been performed due to a lack of genetic and virological data for most members of this family. To elucidate the molecular determinants responsible for human pathogenic bunyavirus virulence and evolution/emergence, we have selected viruses from the genus Orthobunyavirus and ungrouped bunyaviruses as research subjects, and are applying three initial approaches/projects (1) Mechanisms of virulence acquisition in Ngari virus (NRIV) (2) Molecular determinants of host range in Simbu serogroup viruses and (3) Molecular characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses potentially causing human disease. The genus Orthobunyavirus is the largest in the Bunyaviridae and contains more than 170 individual viruses divided into 18 serogroups. These viruses are distributed worldwide and found in a broad range of ecological zones due to the variety of non-vertebrate and vertebrate species able to serve as vectors/reservoirs. Orthobunyaviruses are arthropod-borne and can be transmitted by mosquitoes, culicoid flies and ticks. They are maintained in nature through an arthropod vector/reservoir cycle as well as a vertebrate host (small mammals, human, bats, etc) amplification cycle. To date, over 35 of these viruses have been identified as human pathogens, with a broad spectrum of associated clinical diseases, including systemic febrile illnesses (with arthritis), encephalitic illnesses of varying severity and hemorrhagic fever. (1) Mechanisms of virulence acquisition in Ngari virus (NRIV): In 1997-98 a large outbreak of Rift Valley fever (RVF) occurred in Kenya and Somalia. During this outbreak, NRIV was also identified as the causative agent of hemorrhagic fever (HF). NRIV was recognized as a naturally occurring genetic reassortant between Bunyamwera virus (BUNV;L and S segments) and Batai virus (BATV;M segment), both of which belong to the bunyamwera serogroup in the genus Orthobunyavirus. Interestingly, both parental viruses cause febrile illness, but not severe HF in humans. This is a perfect example of the important role that genetic reassortment plays in the evolution of viruses and of the resulting changes in virulence that can result. Therefore, we are using NRIV as a model to understand the molecular mechanisms underlying the emergence of novel pathogenic bunyaviruses in nature. As a starting point we are carrying out full genome sequencing analysis for 3 NRIV (genotype: L-BUNV/M-BATV/S-BUNV), 5 BUNV and 5 BATV isolates. Phylogenetic analysis for L, M and S segments revealed branching inconsistencies even among BUNVs and suggests the existence of reassortants possessing the genotype L-undefined/M-BUNV/S-BATV. This novel BUNV/BATV reassortant may possess human pathogenic potential. Based on this observation, we are now expanding our genome sequence analysis to include other orthobunyaviruses isolated in Africa in order to better define the genetic relationships among African bunyaviruses. In addition, we have started to develop molecular biological tools including reverse genetics systems to generate recombinant NRIV, BUNV and BATV from cDNA and thus allow us to investigate molecular determinants of NRIV virulence in future. (2) Molecular determinants of host range in Simbu serogroup viruses: The simbu serogroup also belongs to the genus Orthobunyavirus and can be categorized into those viruses that infect i) humans (Oropouche virus), ii) livestock animals (e.g., Akabane virus) or iii) vertebrate hosts (rodents, birds, monkey etc) other than humans or livestock (e.g, Mermet virus). Among the viruses of the simbu serogroup, Oropouche virus (OROV) is unique in causing human disease, which is clinically characterized as an acute febrile disease (Oropouche fever). Since its discovery, OROV has caused more than 30 outbreaks (resulting in at least 500,000 cases between 1960 and 2009) in Brazil, Peru, Panama, and Trinidad and Tobago. It is currently unknown why OROV causes disease in human, but other simbu viruses do not. In order to identify the viral determinants of host range among simbu serogroup viruses, we will compare the genetic and virological characteristics of OROV and other related viruses, especially those that infect non-livestock animals, since these viruses are genetically closer to OROV than those that infect livestock. As an initial step, we are carrying out analysis of the full genome sequence of over 30 simbu serogroup viruses in order to define their genetic relationship. Our analyses have revealed genetic diversity among OROVs isolated from human cases. In addition, since OROV is an important pathogen that has public health significance in South America, we have started to develop reverse genetics system for OROV to study its molecular biology and pathogenesis. (3) Molecular characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses potentially causing human disease: Despite the discovery and isolation of numerous bunyaviruses potentially causing disease in humans and/or animals, genetic information regarding these viruses is limited. This has hampered our understanding of the evolution and ecology/epidemiology of these viruses as well as the development of diagnostics tools, making these bunyavirus infections neglected infectious diseases. In order to better understand the relationships between the molecular biological characteristics of uncharacterized viruses and their zoonotic potential, as well as their evolution, we will conduct an extensive genetic analysis and biological characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses isolated from Africa, Asia, South and North America. Currently, we are preparing the materials for the genetic characterization of several such uncharacterized viruses.